A new record: The largest chemically synthesized polypeptide composed of 304 residues, which corresponds to folded K48‐tetraubiquitin, has been achieved (see scheme). The presented synthetic method could be applied to any of the remaining tetraubiquitin chains, and should ultimately assist ongoing efforts to unravel how the remarkable diversity of ubiquitin signaling is achieved.
Neuer Rekord: Das größte chemisch synthetisierte Polypeptid besteht aus 304 Resten und entspricht gefaltetem K48‐Tetraubiquitin (siehe Schema). Da die Synthesemethode auch auf die übrigen Tetraubiquitinketten angewendet werden kann, könnte sie auf lange Sicht dabei helfen, die bemerkenswerte Vielfalt der Ubiquitin‐Signalwege aufzuklären.
Ubiquitination is a key posttranslational modification, which affects numerous biological processes and is reversed by a class of enzymes known as deubiquitinases (DUBs). This family of enzymes cleaves mono-ubiquitin or poly-ubiquitin chains from a target protein through different mechanisms and mode of interactions with their substrates. Studying the role of DUBs in health and diseases has been a major goal for many laboratories both in academia and in industry. However, the field has been challenged by the difficulties in obtaining native substrates and novel reagents using traditional enzymatic and molecular biology approaches. Recent advancements in the synthesis and semisynthesis of proteins made it possible to prepare several unique ubiquitin conjugates to study various aspects of DUBs such as their specificities and structures. Moreover, these approaches enable the preparation of novel activity based probes and assays to monitor DUB activities in vitro and in cellular contexts. Efforts made to bring new chemical entities for the selective inhibition of DUBs based on these tools are also highlighted with selected examples.
Ubiquitination-the attachment of ubiquitin to a protein target-is involved in a wide range of cellular processes in eukaryotes. This dynamic posttranslational modification utilizes three enzymes to link, through an isopeptide bond, the C-terminal Gly of ubiquitin to the lysine side chain from a protein target. Progress in the field aiming at deciphering the role of ubiquitination in biological processes has been very dependent on the discovery of the enzymatic machinery, which is known to be very specific to each protein target. Chemical approaches offer a complementary route to the biochemical methods to construct these conjugates in vitro in order to assist in unraveling the role of ubiquitination on protein function. Herein is presented a novel method for the rapid synthesis of ubiquitinated peptides employing solid-phase peptide to generate the critical isopeptide linkage. Using these tools, several ubiquitinated peptides derived from known ubiquitinated proteins were prepared. Among them is the ubiquitinated C-terminal fragment of H2B, which can be used in the synthesis of monoubiquitinated H2B. For the first time, we systematically assessed the effect of the length of the ubiquitinated peptides on the UCH-L3 activity and found that peptides of up to ∼20 residues are preferred substrates.
Ubiquitination/ubiquitylation is involved in a wide range of cellular processes in eukaryotes, such as protein degradation and DNA repair. Ubiquitination is a reversible post-translational modification, with the removal of the ubiquitin (Ub) protein being catalyzed by a family of enzymes known as deubiquitinases (DUBs). Approximately 100 DUBs are encoded in the human genome and are involved in a variety of regulatory processes, such as cell-cycle progression, tissue development, and differentiation. DUBs were, moreover, found to be associated with several diseases and as such are emerging as potential therapeutic targets. Several directions have been pursued in the search for lead anti-DUB compounds. However, none of these strategies have delivered inhibitors reaching advanced clinical stages due to several challenges in the discovery process, such as the absence of a highly sensitive and practically available high-throughput screening assay. In this study, we report on the design and preparation of a FRET-based assay for DUBs based on the application of our recent chemical method for the synthesis of Ub bioconjugates. In the assay, the ubiquitinated peptide was specifically labeled with a pair of FRET labels and used to screen a library comprising 1000 compounds against UCH-L3. Such analysis identified a novel and potent inhibitor able to inhibit this DUB in time-dependent manner with k(inact) = 0.065 min(-1) and K(i) = 0.8 μM. Our assay, which was also found suitable for the UCH-L1 enzyme, should assist in the ongoing efforts targeting the various components of the ubiquitin system and studying the role of DUBs in health and disease.
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